This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The overall aim of this research program is to determine how [NiFe]-hydrogenase enzymes are made in bacteria. These enzymes are found in many microorganisms, including Escherchia coli (E. coli) and the pathogenic bacteria Helicobacter pylori, and they are key components in the metabolic pathways that either make hydrogen gas or use it as an energy source. The enzyme has a complicated catalytic active site that contains nickel and iron bound to the protein in addition to several non-protein ligands. It is known that the biosynthesis of hydrogenases requires the coordinated activity of multiple helper proteins to deliver all of the components and assemble the metallocenter correctly. However, very little is known about the mechanisms of action of the individual proteins, how they interrelate with each other, how metal is transferred, or how metal selectivity is achieved. An understanding of this multi-step process is essential in order to realize the potential of hydrogenase enzymes for biotechnology and consumable energy applications, or to evaluate the component proteins as antibiotic targets. Furthermore, this study will contribute to our knowledge about intracellular transition metal homeostasis, a fundamental aspect of life. This experiments described in this proposal are designed to elucidate the molecular details of hydrogenase biosynthesis with a focus on the proteins that insert the nickel into the enzyme center. The proposed work is a study of the nickel insertion proteins by using X-ray absorption spectroscopy (XAS). The first experiments will provide high-resolution data about the coordination spheres of the protein metal-binding sites. We will then use XAS on multi-component samples to determine how the metal-binding sites change when the proteins interact with each other and additional cofactors. This research program will contribute to our understanding of the mechanisms of the hydrogenase biosynthetic pathway.